In numerous cases of surgical intervention, it is necessary to replace, complete or strengthen missing or injured tissues. No ideal substitute for connective tissue replacement is currently available. It is known that collagen modifies the morphology, migration, adhesion and, in some cases, the differentiation and growth of cells.sup.1,2. Different types of collagen (types I, III and IV), gelatin, purified or reconstituted extracellular matrices, glycoproteins such as fibronectin, laminin and vitronectin, fibrinogen/fibrin, composite support containing glycosaminoglycans, basement membrane (Matrigel.TM.), cellulose, chitosan and chitin derivatives can be used to support cell growth.sup.3, and may be used alone or in combination in the porous matrix of the present invention.
To assist wound healing, biocompatible and biodegradable collagen materials may be used. Controlling the rate of biodegradation by crosslinking such implants not only determines the lifetime of the material after application, but may also determine the rate of tissue regeneration.sup.4.
Crosslinking of collagen can be effectively achieved by chemical, radiation and/or dehydrothermal methods. Chemical agents such as glutaraldehyde, hexamethylene-diisocyanate, acyl azide, 1-ethyl -3-(3-dimethylaminopropyl) carbodiimide (EDCI) hydrochloride, N-hydroxysuccinimide, or polyepoxy fixative have been broadly used to create the biological stability of collagen.sup.4-8. Glutaraldehyde is however an example of crosslinking agents having the disadvantage of releasing toxic components during in vivo degradation.sup.32. In addition, the introduction of crosslinking agents to the natural crosslinking of collagen can modify the biological properties of collagen.
As previously demonstrated by co-inventor and others.sup.9-12, gamma or electron beam irradiation has been used to stabilize the collagen structure with beneficial results in clinical applications. Besides, ionizing radiation has been successfully used for grafting collagen onto synthetic materials as polyester vascular prostheses for angiosurgery.sup.13 as well as for the preparation of collagen substitutes such as abdominal paries and dura mater.sup.11.
Synthetic polymers have been used to coat surfaces, including biopolymers surfaces or have been conjugated to biopolymers to modify their properties, for various purposes. Polyethyleneglycols (PEGs) which are versatile polymers having amphiphilic (hydrophilic and hydrophobic) properties.sup.33, are used to increase the resistance of proteins to proteolytic degradation. PEGs have been reported to abrogate the immunogenicity of proteins while preserving their biological properties.sup.34, 35. PEGs were conjugated to various proteins or lipids to produce various delivery systems for drugs, cytokines and enzymes.sup.33,36-39. Using PEG-modified adenosine deaminase, children who have an adenosine deaminase deficiency were successfully treated.sup.36. Neither toxic effect nor hypersensitivity reactions were observed. Furthermore, PEGs were conjugated to liposomes significantly prolong their biological properties.sup.40. More recently, PEGs have been conjugated to pepsinized collagen in solution utilizing succinic anhydride and glutarate reactions.sup.14,15,35.
Previously, only chemically activated PEGs in collagen modification was studied.sup.14,15. Thus, it was found that PEG-collagen derivatives present more favourable biological properties than non-grafted ones. PEG has been also used as plasticizer of collagen and to enhance resorption and protection of peptide drug.sup.16,17. Furthermore, poly(2-hydroxyethyl methacrylate) (pHEMA) has been chemically bound to soluble or insoluble collagen crosslinked by glutaraldehyde.sup.18-22. These polymers are stable against biodegradation and have shown a good biocompatibility without any cytotoxicity.sup.22. Moreover, a great deal of work has been devoted to the use of PEG and HEMA to create synthetic polymers or graft copolymers. Comprehensive reviews of this literature have been published.sup.23-25.
Recently, we have applied chemical or radiation processing to develop a new family of collagen-based biopolymers. Subsequent modification of collagen is performed by systematically varying the grafting solution compositions using hydrophilic and hydrophobic macromonomers with different chain lengths. These specific chemical- or radiation- modified composite materials could be selected to fulfill special biological and medical needs.
It will be therefore appreciated that composite collagen material containing different proportions of synthetic polymers and/or monomers and having different biological properties can be obtained by using chemical or radiation techniques.
The U.S. Pat. No. 4,840,851 describes the use of PEG in the coating of surfaces as protein-repellent, by keeping a freely movable polyethylene oxide chain, unsaturated, and which will not take part in the crosslinking; this part of the PEG derivative molecules is usually a conventional etherified end. The other end is the OH-terminus esterified with a compound having an ethylenically unsaturated group. Radiation has been used and crosslinking is achieved by allylic, acrylic or methacrylic groups. No suggestion is made in this patent of a porous collagen product which, when impregnated with PEG and/or PHEMA and irradiated, has a stable porosity.
The U.S. Pat. No. 4,871,490 describes hydrogels made by mixing natural and synthetic polymers, and optionally a plasticizing agent, in water, this mixture being poured in a mould and cured. No porous material is suggested. The making of a composite collagen sponge cannot be deduced from this reference because curing such a mixture will not result in a porous structure but in a compact one. Therefore, there is no suggestion of impregnating a collagen sponge with PEGs which will produce a stabilized porous material by chemical or radiation methods.
The U.S. Pat. No. 4,978,298 teaches a collagen sponge which is crosslinked with carbodiimide. The composite sponges do not lose the biological properties of collagen (and of added connective tissue factor) and show a decreased inflammatory response. Even though the composite sponges are capable of supporting ingrowth of fibroblasts, there is no indication of a bio-stable porosity.
The U.S. Pat. No. 5,290,548 teaches the coating of implants which precludes adhesion and spreading of cells and will not encourage colonization by infiltration and fixation of cells.
The U.S. Pat. No. 5,162,430 teaches collagen-polymer conjugates. A polymer like MPEG is activated by reaction with a linking group and such an activated polymer is then reacted with the free amino groups of collagen. The conjugated material is taught as being useful as an implant having an increased tensile strength as well as a longer residence time in the body than a non-conjugated crosslinked collagen implant. This reference does not exclude the use of fibrillar collagen and does not exclude colonization of implants by cells. However, this reference does not teach a composite collagen sponge having a stable porosity.
The patent publication EP 568,334 teaches collagen sponges made by soaking gelatin sponges in a mixture of collagen and of a pharmacologically active ingredient enhancing and promoting wound healing. No sponge made of a composite collagen-polymer material and having a stable porosity is neither disclosed nor suggested in this reference.
In tissue repair mechanism, e.g. after wounding or surgery, the cells contributing to new tissue reconstruction need a matrix supporting cell growth. Sponges made of collagen are provided as matrices supporting cell growth. However the pores of these sponges through which fibroblasts infiltrate are prone to collapse shortly after their implantation. Indeed the pores do not remain stable for a time sufficient for fibroblast colonization.
Therefore, there is clearly a need for sponges containing biopolymers capable of supporting cell growth which have a stabilized porosity, allowing for colonization of fibroblasts, as well as an improved resistance to proteolysis.